Interfacial Electromigration for Analysis of Biofluid Lipids in Small Volumes

Lipids are important biomarkers within the field of disease diagnostics and can serve as indicators of disease progression and predictors of treatment effectiveness. Although lipids can provide important insight into how diseases initiate and progress, mass spectrometric methods for lipid characterization and profiling are limited due to lipid structural diversity, particularly the presence of various lipid isomers. Moreover, the difficulty of handling small-volume samples exacerbates the intricacies of biological analyses. In this work, we have developed a strategy that electromigrates a thin film of a small-volume biological sample directly to the air–liquid interface formed at the tip of a theta capillary. Importantly, we seamlessly integrated in situ biological lipid extraction with accelerated chemical derivatization, enabled by the air–liquid interface, and conducted isomeric structural characterization within a unified platform utilizing theta capillary nanoelectrospray ionization mass spectrometry, all tailored for small-volume sample analysis. We applied this unified platform to the analysis of lipids from small-volume human plasma and Alzheimer’s disease mouse serum samples. Accelerated electro-epoxidation of unsaturated lipids at the interface allowed us to characterize lipid double-bond positional isomers. The unique application of electromigration of a thin film to the air–liquid interface in combination with accelerated interfacial reactions holds great potential in small-volume sample analysis for disease diagnosis and prevention.


S1. Electromigration of a thin film in a theta capillary
Lipid standards (PC(18:1/18:1) and PC(16:0/18:1)) were prepared in ACN and diluted to a concentration of 100 µM.PC(18:1/18:1) was loaded into one barrel along with a Pt electrode, and PC(16:0/18:1) was loaded into the other barrel.Upon the application of voltage tuned between 2.8-3.2kV, we were able to observe in the mass spectrometer a high abundance of the PC(16:0/18:1) standard in the barrel without the electrode.Figure S1 shows the migration of solution from the barrel without an electrode to the meniscus of the barrel with the electrode and the importance of the electrode placement.

S2. Volume of thin film migrated via electromigration
Lipid standards (PC 18:1_18:1 and PC 16:0_18:1) were respectively prepared in ACN and diluted to a concentration of 100 µM.PC 18:1_18:1 was loaded into one barrel with a Pt electrode, and PC 16:0_18:1 was loaded into the other barrel.Upon the application of voltage tuned between 2.8-3.2kV, we were able to observe the time for a thin film to migrate from the barrel without the electrode to the barrel with the electrode in the extracted chromatogram of PC 16:0_18:1 (Figure S2).The volume of thin film migration was calculated to be 4.64±1.0nL (Table S2) based on the spray flow rate which was determined to be 103.17±22.45nL/min (Table S1).

S3. Tracking liquid flow in electromigration using the dye Thioflavin S
Thioflavin S at a concentration of 0.7% diluted in water was loaded into one barrel of a 10 µm theta tip emitter and into one barrel of an 80 µm theta tip emitter (Figure S3a and c).Voltage was then applied starting at 0.1 kV and increased in 0.1 kV increments until a voltage of 2.8 kV and 4 kV was achieved for the small and large emitters, respectively.After the application of voltage to the smaller orifice emitter (10 µm), the electroosmosis phenomenon was observed (Figure S3b); when voltage was applied to one barrel higher than the other, the solution in the barrel with the higher voltage migrated to the side with the lower voltage (J.Mass Spectrom.2015, 50 (9), 1063-1070).However, when this same experiment was performed using a large orifice theta tip emitter, a different phenomenon was observed (Figure S3d).Using the large orifice theta tip emitter, we observed electromigration where the solution with the lower voltage (in our experiment, the lower voltage barrel received no voltage) migrated to the barrel to which the voltage is applied.

S4. In situ extraction of lipids from pooled normal human plasma (Innovative Research, Inc.) via electromigration
Serum (0.1 µL) was pipetted onto a glass microscope slide.The theta capillary was then carefully placed on the droplet of serum and a small amount was placed in the first barrel of the theta emitter via capillarity.The second barrel was then loaded with a modified Matyash solution, which contained MTBE and an ACN/H2O (4:1, v/v) solution with 10 mM of NH4Cl, 1mM HCl in a 10:1 volume ratio, and a Pt wire.In-situ lipid extraction was subsequently performed followed by characterization using mass spectrometry.S3 and S4 for lipid identification).

S5. Characterization of fatty acids at the isomer level by electroepoxidation in the interfacial microreactor after electromigration S5.1 Determination of detection limit using electromigration in a theta capillary nESI
Lipid standard solutions (PC(18:1/18:1), m/z 786 with 0.01% formic acid) at 50 µM, 50 nM, 50 pM, 50 fM, and 10 fM were prepared.Samples were loaded using the same solution into a single barrel nESI emitter (>10 µm orifice size) for comparison to the sample loaded into one barrel of the theta emitter.For the comparison, one barrel of the theta emitter was loaded with the same solution as the single barrel nESI emitter, and the other barrel was loaded with ACN:H2O (v/v=4:1).Lipid was detected at 10 fM using electromigration with the theta emitter.In comparison, the lipid was not detected using a single barrel nESI emitter at this concentration.

S5.2 Characterization of fatty acids at the isomer level by electroepoxidation in the interfacial microreactor after electromigration
The theta capillary was placed in front of the MS inlet and a Pt wire was positioned in a barrel containing EtOAC with 10 mM NH4Cl and 1 mM HCl (Barrel 1).The Pt electrode acts as the working electrode able to provide electrical contact with the spray solvent upon the application of voltage.To initiate electroepoxidation, the voltage was tuned between 2.8-3.2kV, which was turned off by applying a voltage above 3.5 kV based on the spray modes that occur in the large orifice theta emitter.When the voltage was tuned between 2.8 and 3.2 kV, a meniscus was formed at the tip of the emitter, which is where the epoxidation reaction was accelerated.Serum (0.1 µL) was applied to a microscope slide, and the theta emitter tip was carefully placed on the droplet.Via capillary action, a small amount of the plasma droplet was taken up by the emitter (barrel 2).

S5.3 Investigation of electromigration of lipids with and without mouse serum matrix
We compared the electromigration of lipids with and without mouse serum matrix.In the study of the lipid electromigration in mouse serum, PC 18:1_18:1 and PC 16:0_18:1 were prepared in ACN:H2O (1:1) solvent and in serum, respectively.PC 18:1_18:1 (10 µL) was loaded into barrel a with the electrode and PC 16:0_18:1 (0.5 µL) in mouse serum was loaded into barrel b without the electrode.Upon the application of voltage to the electrode, we observed a 36 seconds-delay in the electromigration of PC 16:0_18:1 in serum.This could be attributed to the fact that serum is more viscous than the organic solvent which results in the slow migration when applying the same force of migration.

S6. Acceleration of epoxidation of negatively charged fatty acids in mouse serum
An external AC wave function generator (Stanford Research Systems, Sunnyvale, CA) was connected to a digital storage oscilloscope (Hantek Electronic Co., Ltd., Qingdao, China) with a power amplifier (Trek, Lockport, NY).Since the epoxidation reaction was initiated with a positive potential, AC voltage was used to epoxidize the negatively charged fatty acids.A square wave function with the offset=0, frequency=50Hz, and amplitude was increased to apply a tuned voltage of 2.8-3.2kV to initiate the migration of serum to the meniscus for the in situ extraction and epoxidation.

S7. Changes of C=C bond positional isomer ratios in the GHS-R knockout 5xFAD mouse serum
We have compared the changes of C=C bond positional isomer ratios in the GHS-R knockout 5xFAD mouse serum compared to those in the normal 5xFAD mouse serum (control).Significant differences were found in the C=C bond positional isomer ratios shown in Figure S9.

S1.
Electromigration of a thin film in a theta capillary S2.Volume of thin film migrated via electromigration S3.Tracking liquid flow in electromigration using the dye Thioflavin S S4.In situ extraction of lipids from pooled normal human plasma (Innovative Research, Inc.) via electromigration S5.Characterization of fatty acids at the isomer level by electroepoxidation in the interfacial microreactor after electromigration S6.Acceleration of epoxidation of negatively charged fatty acids in mouse serum S7.Changes of C=C bond positional isomer ratios in the GHS-R knockout 5xFAD mouse serum S-2

Figure S2 .
Figure S2.Extracted ion chromatogram for the time measurements used to calculate the volume of thin film migrated via electromigration.

Figure S4 .
Figure S4.Mass spectrum of human plasma lipids after electromigration and an in situ modified Maytash extraction in a large-orifice theta capillary coupled with mass spectrometry analysis (See TableS3and S4 for lipid identification).

Figure S6 .
Figure S6.Full mass spectrum showing both protonated and epoxidated FA ions.